This invention relates to the composition and the utility of substituted naphthyl benzofuran derivatives as inhibitors of plasminogen activator inhibitor-1 (PAI-1) and as therapeutic compositions for treating conditions resulting from fibrinolytic disorders such as deep vein thrombosis and coronary heart disease, and pulmonary fibrosis.
Plasminogen activator inhibitor-1 (PAI-1) is a major regulatory component of the plasminogen-plasmin system. PAI-1 is the principal physiologic inhibitor of both tissue type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA). Elevated plasma levels of PAI-1 have been associated with thrombotic events as indicated by animal experiments (Krishnamurti, Blood, 69, 798 (1987); Reilly, Arteriosclerosis and Thrombosis, 11, 1276 (1991); Carmeliet, Journal of Clinical Investigation, 92, 2756 (1993)) and clinical studies (Rocha, Fibrinolysis, 8, 294, 1994; Aznar, Haemostasis 24, 243 (1994)). Antibody neutralization of PAI-1 activity resulted in promotion of endogenous thrombolysis and reperfusion (Biemond, Circulation, 91, 1175 (1995); Levi, Circulation 85, 305, (1992)). Elevated levels of PAI-1 have also been implicated in diseases of women such as polycystic ovary syndrome (Nordt, Journal of clinical Endocrinology and Metabolism, 85, 4, 1563 (2000)) and bone loss induced by estrogen deficiency (Daci, Journal of Bone and Mineral Research, 15, 8, 1510 (2000)). Accordingly, agents that inhibit PAI-1 would be of utility in treating conditions originating from fibrinolytic disorder such as deep vein thrombosis, coronary heart disease, pulmonary fibrosis, polycystic ovary syndrome, etc.
U.S. Pat. No. 6,110,963 claims benzofuran derivatives useful in the treatment of hyperglycemia.
WO 95/10513 (Pfizer Inc.) discloses benzothiophenes and related compounds of formula I as estrogen agonists. 
wherein: X=S, O, etc.; Y=alkyl, cycloalkyl, cycloalkenyl, phenyl, a 5- or 6-membered heterocycle, or a bicyclic ring system consisting of a 5- or 6-membered heterocyclic ring fused to a phenyl ring, all optionally substituted.
U.S. Pat. Nos. 5,948,795 and 5,962,698 (Eli Lilly and Company) describe benzothiophene derivatives of formula I and their use as PAI-1 inhibitors. 
wherein: R1, R2, and R3 are independently xe2x80x94OH, xe2x80x94OCO(C1-C6)alkyl, xe2x80x94O(CO)O(C1-C6)alkyl, xe2x80x94OCO-phenyl, xe2x80x94OCO-substituted phenyl, or O(CO)O-phenyl; and R4 is N-pyrrolidinyl, N-piperidinyl, or N-hexamethyleneimino.
EP 0 655 439 (Eli Lilly and Company) teaches 5,6 fused ring bicyclic compounds inclusive of indoles, benzofurans, and benzothiophenes corresponding to the general formula I as platelet aggregation inhibitors: 
wherein: A is an acidic group linked to the 5-membered ring by linking group La, where La is either a bond or a divalent chain of 1-15 carbon atoms; B is a basic group linked to the 6-membered ring by linking group Lb, where Lb is either a bond or a divalent chain of 1-15 carbon atoms.
This invention comprises compounds of formula 1:
wherein:
R, R1, R2, and R3 are independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, xe2x80x94CH2-C3-C6 cycloalkyl), C1-C6 alkanoyl, halo, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, perfluoroalkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, amino, xe2x80x94NH(alkyl of 1-6 carbon atoms), xe2x80x94N(alkyl of 1-6 carbon atoms)2, and perfluoroalkoxy of 1-6 carbon atoms;
R4 is hydrogen, alkyl of 1-6 carbon atoms, branched alkyl of 1-6 carbon atoms, perflouroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkenyl-aryl, xe2x80x94CH2R5, xe2x80x94CH(OH)R5, xe2x80x94C(O)R5, xe2x80x94CH(SH)R5, or xe2x80x94C(S)R5 or xe2x80x94(CH2)nxe2x80x94C3 to C6 cycloalkyl wherein n is an integer of from 0 to 2;
R5 is hydrogen, alkyl of 1-6 carbon atoms, branched alkyl of 1-6 carbon atoms, perflouroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkenyl-aryl or xe2x80x94(CH2)nxe2x80x94C3 to C6 cycloalkyl wherein n is an integer of from 0 to 2;
R6 is selected from hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, xe2x80x94CH2-cycloalkyl of 3 to 6 carbon atoms, alkylaryl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
n is an integer of 0-6;
A is COOH, or an acid mimic or mimetic; or a pharmaceutically acceptable salt or ester form thereof.
Acid mimic or mimetics which are included in the acidic groups of this invention, as noted in the definition of A, above, particularly include the pharmaceutically useful carboxylic acid mimics or mimetics known in the art, such as those described in R. Silverman, The Organic Chemistry of Drug Design and Drug Action, Academic Press (1992), the contents of which are incorporated herein by reference. Non-limiting examples of these acid mimics include such as tetrazole, SO3H, PO3H2, tetronic acid, etc., or groups having the formulae: 
wherein R7 is C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, xe2x80x94CH2xe2x80x94(C3-C6 cycloalkyl), C3-C6 cycloalkenyl, xe2x80x94CH2xe2x80x94(C3-C6 cycloalkenyl), optionally substituted aryl or heteroaryl groups or optionally substituted xe2x80x94C1-C6 alkyl-aryl or xe2x80x94C1-C6 alkyl-heteroaryl, with the aryl and heteroaryl groups and their optional substitution as defined herein.
A subset of the compounds of this invention are those of the formula 2:
wherein R, R1, R2, R3, R4, R6, A, and n are as defined above, or a pharmaceutically acceptable salt or ester form thereof.
A further subset of the compounds of this invention comprises those having the formula 3: 
wherein R, R1, R2, and R3, are as defined above,
n=0
A1 is a carboxylic acid or a tetrazole group2 
R6 is a hydrogens, C1-C6 alkyl or a benzyl group optionally substituted by from 1 to 3 groups selected from the list of substituents for the aryl or heteroaryl groups described below;
Y represents two single bonded H atoms; one H and one OH; or a double bonded oxygen atom; and
R5 is selected from C1-C8 alkyl, preferably C1-C6 alkyl, C3-C6 cycloalkyl, xe2x80x94CH2xe2x80x94C3-C6 cycloalkyl, or benzyl, the rings of the cycloalkyl and benzyl groups being optionally substituted by from 1 to 3 groups selected from halogen, C1-C3 alkyl, C1-C3 perfluoroalkyl, preferably xe2x80x94CF3, xe2x80x94Oxe2x80x94C1-C3 perfluoroalkyl, preferably xe2x80x94Oxe2x80x94CF3, C1-C3 alkoxy, xe2x80x94OH, xe2x80x94NH2, or xe2x80x94NO2;
or a pharmaceutically acceptable salt or ester form thereof.
As used herein, xe2x80x9carylxe2x80x9d refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl). Preferred aryl groups include phenyl, naphthyl and the like. As used herein, xe2x80x9cheteroarylxe2x80x9d refers to a monocyclic or bicyclic aromatic group of from 1 to carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring). Such heteroaryl groups can have a single ring, such as pyridyl, pyrrolyl or furyl groups, or multiple condensed rings, such as indolyl, indolizinyl, benzofuranyl or benzothienyl groups. Preferred heteroaryls include pyridyl, pyrrolyl and furyl. It will be understood that the definitions of aryl and heteroaryl also refer to those portions of any aroyl or heteroaroyl groups described herein.
Unless otherwise limited by the definition for the aryl or heteroaryl groups herein, such groups can optionally be substituted with from 1 to 5 substituents selected from the group consisting of acyloxy, hydroxy, acyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, amino, amino substituted by one or two alkyl groups of from 1 to 6 carbon atoms, aminoacyl, acylamino, azido, cyano, halo, nitro, thioalkoxy of from 1 to 6 carbon atoms, substituted thioalkoxy of from 1 to 6 carbon atoms, and trihalomethyl. Substituents on the alkyl, alkenyl, alkynyl, thioalkoxy and alkoxy groups mentioned above include halogens, CN, OH, and amino groups. Preferred substituents on the aryl groups herein include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.
The preferred salt forms of the compounds herein include but are not limited to sodium salts, and potassium salts. Other useful salt forms of these compounds include those formed with pharmaceutically acceptable inorganic and organic bases known in the art. Salt forms prepared using inorganic bases include hydroxides, carbonates or bicarbonates of the therapeutically acceptable alkali metals or alkaline earth methals, such as sodium potassium, magnesium, calcium and the like. Acceptable organic bases include amines, such as benzylzmine, mono-, di- and trialkylamines, preferably those having alkyl groups of from 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, mono-, di-, and triethanolamine. Also useful are alkylene diamines containing up to 6 carbon atoms, such as hexamethylenediamine; cyclic saturated or unsaturated bases containing up to 6 carbon atoms, including pyrrolidine, peperidine, morpholine, piperazine and their N-alkyl and N-hydroxyalkyl derivatives, such as N-methyl-morpholine and N-(2-hyroxyethyl)-piperidine, or pyridine. Quaternary salts may also be formed, such as tetralkyl forms, such as tetramethyl forms, alkyl-alkanol forms, such as methyl-triethanol or trimethyl-monoethanol forms, and cyclic ammonium salt forms, such as N-methylpyridinium, N-methyl-N-(2-hydroxyethyl)-morpholinium, N,N-di-methylmorpholinium, N-methyl-N-(2-hydroxyethyl)-morpholinium, or N,N-dimethyl-piperidinium salt forms. These salt forms may be prepared using the acidic compound(s) of Formula I and procedures known in the art.
Ester forms of the compounds of this invention include straight chain alkyl esters having from 1 to 6 carbon atoms or branched chain alkyl groups containing 3 or 6 carbon atoms, including methyl, ethyl, propyl, butyl, 2-methylpropyl and 1,1-dimethylethyl esters. Other esters useful with this invention include those of the formula xe2x80x94COOR5 wherein R5 is selected from the formulae: 
wherein R11, R12, R13, R14 are independently selected from hydrogen, alkyl of from 1 to 10 carbon atoms, aryl of 6 to 12 carbon atoms, arylalkyl of from 6 to 12 carbon atoms; heteroaryl or alkylheteroaryl wherein the heteroaryl ring is bound by an alkyl chain of from 1 to 6 carbon atoms.
Among the preferred ester forms of the compounds herein include but not limited to C1-C6 alkyl esters, C3-C6 branched alkyl esters, benzyl esters, etc.
As used herein, xe2x80x9carylxe2x80x9d refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl). Preferred aryl groups include phenyl, naphthyl and the like. As used herein, xe2x80x9cheteroarylxe2x80x9d refers to a monocyclic or bicyclic aromatic group of from 1 to carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring). Such heteroaryl groups can have a single ring, such as pyridyl, pyrrolyl or furyl groups, or multiple condensed rings, such as indolyl, indolizinyl, benzofuranyl or benzothienyl groups. Preferred heteroaryls include pyridyl, pyrrolyl and furyl.
Unless otherwise limited by the definition for the aryl or heteroaryl groups herein, such groups can optionally be substituted with from 1 to 5 substituents selected from the group consisting of acyloxy, hydroxy, acyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, amino, amino substituted by one or two alkyl groups of from 1 to 6 carbon atoms, aminoacyl, acylamino, azido, cyano, halo, nitro, thioalkoxy of from 1 to 6 carbon atoms, substituted thioalkoxy of from 1 to 6 carbon atoms, and trihalomethyl. Substituents on the alkyl, alkenyl, alkynyl, thioalkoxy and alkoxy groups mentioned above include halogens, CN, OH, and amino groups. Preferred substituents on the aryl groups herein include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.
The compounds of the present invention are inhibitors of the serine protease inhibitor PAI-1, and are therefore useful in the treatment, inhibition, prevention or prophylaxis in a mammal, preferably in a human, of those processes which involve the production and/or action of PAI-1. Thus, the compounds of the invention are useful in the treatment or prevention of noninsulin dependent diabetes mellitus and cardiovascular disease caused by such condition, and prevention of thrombotic events associated with coronary artery and cerebrovascular disease. These compounds would also be useful for inhibiting the disease process involving the thrombotic and prothrombotic states which include, but are not limited to, formation of atherosclerotic plaques, venous and arterial thrombosis, myocardial ischemia, atrial fibrillation, deep vein thrombosis, coagulation syndromes, pulmonary fibrosis, cerebral thrombosis, thromboembolic complications of surgery (such as joint replacement), and peripheral arterial occlusion. These compouds are also useful in treating stroke associated with or resulting from atrial fibrillation.
The compounds of the invention may also be used in the treatment of diseases associated with extracellular matrix accumulation, including, but not limited to, renal fibrosis, chronic obstructive pulmonary disease, polycystic ovary syndrome, restenosis, renovascular disease and organ transplant rejection.
The compounds of the invention may also be useful in the treatment of malignancies, and diseases associated with neoangiogenesis (such as diabetic retinopathy).
The compounds in the invention may also be used in conjunction with and following processes or procedures involving maintaining blood vessel patency, including vascular surgery, vascular graft and stent patency, organ, tissue and cell implantation and transplantation. The compounds in the invention may also be useful in the treatment of inflammatory diseases, septic shock and the vascular damage associated with infections.
The compounds of the invention are useful for the treatment of blood and blood products used in dialysis, blood storage in the fluid phase, especially ex vivo platelet aggregation. The present compounds may also be added to human plasma during the analysis of blood chemistry in hospital settings to determine the fibrinolytic capacity thereof.
The compounds in the present invention may also be used in combination with prothrombolytic, fibrinolytic and anticoagulant agents.
The compounds of the present invention may also be used to treat cancer including, but not limited to, breast and ovarian cancer, and as imaging agents for the identification of metastatic cancers.
The compounds of the invention may also be used in the treatment of Alzheimer""s disease. This method may also be characterized as the inhibition of plasminogen activator by PAI-1 in a mammal, particularly a human, experiencing or subject to Alzheimer""s disease. This method may also be characterized as a method of increasing or normalizing levels of plasmin concentration in a mammal, particularly those experiencing or subject to Alzheimer""s disease.
The compounds of the invention may be used for the treatment of myelofibrosis with myeloid metaplasia by regulating stromal cell hyperplasia and increases in extracellular matrix proteins.
The compounds of the invention may also be used in conjunction with protease inhibitor-containing highly active antiretroviral therapy (HAART) for the treatment of diseases which orginate from fibrinolytic impairment and hyper-coagulability of HIV-1 infected patients receiving such therapy.
The compounds of the invention may be used for the treatment of diabetic nephropathy and renal dialysis associated with nephropathy.
The compounds of the invention may be used to treat cancer, septicemia, obesity, insulin resistance, proliferative diseases such as psoriasis, improving coagulation homeostasis, cerebrovascular diseases, microvascular disease, hypertension, dementia, osteoporosis, arthritis, asthma, heart failure, arrhythmia, angina, and as a hormone replacement agent, treating, preventing or reversing progression of atherosclerosis, Alzheimer""s disease, osteoporosis, osteopenia; reducing inflammatory markers, reducing C-reactive protein, or preventing or treating low grade vascular inflammation, stroke, dementia, coronary heart disease, primary and secondary prevention of myocardial infarction, stable and unstable angina, primary prevention of coronary events, secondary prevention of cardiovascular events, peripheral vascular disease, peripheral arterial disease, acute vascular syndromes, reducing the risk of undergoing a myocardial revascularization procedure, microvascular diseases such as nephropathy, neuropathy, retinopathy and nephrotic syndrome, hypertension, Type 1 and 2 diabetes and related diseases, hyperglycemia, hyperinsulinemia, malignant lesions, premalignant lesions, gastrointestinal malignancies, liposarcomas and epithelial tumors, proliferative diseases such as psoriasis, improving coagulation homeostasis, and/or improving endothelial function, and all forms of cerebrovascular diseases.
The compounds of the invention may be used for the topical applications in wound healing for prevention of scarring.
This invention also comprises methods for the treatment, inhibition, prevention or prophylaxis in a mammal of each of the conditions or maladies listed herein. Each method comprises administering to a mammal in need thereof a pharmaceutically or therapeutically effective amount of a compound of this invention, or a pharmaceutically acceptable salt or ester form thereof.
This invention also provides pharmaceutical compositions comprising a pharmaceutically or therapeutically effective amount of a compound of this invention, or a pharmaceutically acceptable salt or ester form thereof, either alone or in combination with one or more pharmaceutically acceptable carriers or excipients (i.e. pharmaceutically acceptable materials with no pharmacological effects). It will be understood that a pharmaceutically or therapeutically effective amount of a compound herein refers to an amount of the compound in question which will sufficiently inhibit the serine protease inhibitor PAI-1 in the mammal in need thereof to a sufficient extent to provide a desirable improvement in the condition in question or provide sufficient inhibition of the serine protease inhibitor PAI-1 to prevent, inhibit or limit the onset of the physiological basis for the malady or condition in question.
The compounds of the present invention can be readily prepared according to the methods described in the following reaction schemes or modification thereof using readily available starting materials, reagents and conventional synthetic procedures. It is also possible to make use of variants of these process steps, which in themselves are known to and well within the preparatory skills of medicinal chemists.
Synthesis of substituted 2-naphthylbenzofurans 9 from the appropriate alkyne 2 and o-halo phenols is described in the literature (Torii, Synlett 1992, (6), 515-516), Scheme I. 
Preparation of 6-bromo-1-chloro-2-naphthol 4 from the corresponding 6-bromo-2-naphthol 3 is described in the literature (Buu-Hoi, JOC 1951, 16, 185), Scheme II. 6-Bromo-1-methyl-2-naphthol 7 was prepared as depicted in reaction Scheme II. Reaction of 6-bromo-2-naphthol 3 with aqueous dimethyl amine and aqueous formaldehyde in alcohol afforded the Mannich product 5. Reaction of 5 with acetyl chloride in methylene chloride or chloroform afforded the acetate 6. Hydride reduction of 6 in a solvent such as ethanol followed by basic work-up yielded the desired 6-bromo-1-methyl-2-naphthol 7. 
In Scheme III, substituted 2-naphthyl benzofurans 9 were prepared by cross-coupling of substituted bromo naphthalenes with various benzofuran boronic acids 8 using standard palladium-catalyzed cross-coupling procedures under basic conditions in a variety of solvents or mixtures of solvents such as dioxane, water, toluene, alcohol, or THF. Reaction of 9 with acid chlorides or acid anhydrides in the presence of a Lewis acid such as tin(IV)chloride was carried out in a solvent such as methylene chloride or chloroform to afford the 3-acyl benzofuran derivatives 10. Conversion of the methoxy group of 10 to the corresponding hydroxy group was accomplished by treatment of 10 with boron trichloride or boron tribromide in methylene chloride affording derivatives 11. Subsequent bromination of 11 using bromine in acetic acid in the presence of sodium acetate furnished the bromo derivatives 12a. Reduction of 12a or 11 with sodium borohydride in a solvent such as ethanol afforded the alcohols 12b or 12c respectively. Further reduction of 12b or 12c with triethylsilane in a solvent such as methylene chloride under acidic conditions (triflouro acetic acid) afforded the alkyl derivatives 12d and 12e respectively. 
In a similar manner, preparation of substituted 2-naphthyl benzofuran derivatives 13a, 13b, 13c, 14a, 14b, and 14c from 6-bromo naphthalenes 4 or 7, was accomplished by following a modification of the reaction sequence described in Scheme III.
In Scheme IV, compounds 12, 13, or 14 were alkylated with bromoacetonitrile using a base such as potassium carbonate or cesium carbonate in a solvent such as acetone to give the nitrites 15. Conversion of the nitrites 15 to the corresponding tetrazole derivatives 1 a was carried out by reacting with sodium azide in the presence of ammonium chloride in a solvent such as DMF at a temperature of 80-100xc2x0 C. Similarly, alkylation of 12, 13, or 14 with a bromoacetate under basic conditions as described above afforded the acetate derivatives 16. Saponification of 16 furnished the corresponding acetic acid derivatives 1b. Alternatively, coupling of compounds 12, 13, or 14 with hydroxy esters such as phenyllactic acid esters under standard Mitsunobu reaction conditions afforded the substituted esters 17. Hydrolysis of the ester as described above afforded the desired acids 1c. 
This invention also provides pharmaceutical compositions comprised of substituted naphthyl benzofuran derivatives (I) either alone or in combination with excipients (i.e. pharmaceutically acceptable materials with no pharmacological effects). Such compositions for treating conditions resulting from fibrinolytic disorder such as deep vein thrombosis and coronary heart disease, pulmonary fibrosis, etc.
The precise dosage to be employed depends upon several factors including the host, whether in veterinary medicine or human medicine, the nature and severity of the condition being treated, the mode of administration and the particular active substance employed. The compounds may be administered by any conventional route, in particular enterally, preferably orally in the form of tablets or capsules.
Administered compounds can be in the free form or pharmaceutically acceptable salt form as appropriate, for use as a pharmaceutical, particularly for use in the prophylactic or curative treatment of atherosclerosis and sequelae (angina pectoris, myocardial infarction, arrhythmias, heart failure, kidney failure, stroke, peripheral arterial occlusion, and related disease states). These measures will slow the rate of progress of the disease state and assist the body in reversing the process direction in a natural manner.
Any suitable carrier known to the art can be used to prepare the pharmaceutical compositions. In such a composition, the carrier may be a solid, liquid or mixture of a solid and a liquid. Solid compositions include powders, tablets and capsules. A solid carrier can be one or more substances which may also act as a flavoring agent, lubricant, solubilizer, suspending agent, binder, or tablet disintegrant. In powders, the carrier is a finely divided solid, which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. Suitable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, hydroxymethyl cellulose, sodium carboxymethyl cellulose, a low melting wax, cocoa butter, and the like. Encapsulating materials may also be employed with the compounds of this invention, and the term xe2x80x9ccompositionxe2x80x9d is intended to include the active ingredient in combination with an encapsulating material as a formulation, with or without other carriers. Cachets may also be used in the delivery of the anti-atherosclerotic medicament of this invention.
Sterile liquid compositions include solutions, suspensions, emulsions, syrups and elixirs. The compounds of this invention may be dissolved or suspended in the pharmaceutically acceptable carrier, such as sterile water, sterile organic solvent or a mixture of both. Preferably the liquid carrier is one suitable for parental injection. Where the compounds are sufficiently soluble they can be dissolved directly in normal saline with or without the use of suitable organic solvents, such as propylene glycol or polyethylene glycol. If desired, dispersions of the finely divided compounds can be made-up in aqueous starch or sodium carboxymethyl cellulose solution, or in a suitable oil, such as arachis oil. Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by intramuscular, intraperitoneal or subcutaneous injection. In many instances a liquid composition form may be used instead of the preferred solid oral method of administration.
It is preferred to prepare unit dosage forms of the compounds for standard administration regimens. In this way, the composition can be subdivided readily into smaller doses at the physicians direction. For example, unit dosages may be made up in packeted powders, vials or ampoules and preferably in capsule or tablet form. The active compound present in these unit dosage forms of the composition may be present in an amount of from about one gram to about fifteen grams or more, for single or multiple daily administration, according to the particular need of the patient. The daily dose of active compound will vary depending upon the route of administration, the size, age and sex of the patient, the severity of the disease state, and the response to the therapy as traced by blood analysis and the patients recovery rate. By initiating the treatment regimen with a minimal daily dose of about one gram, the blood levels of PAI-1 and the patients symptomatic relief analysis may be used to determine whether a larger dose is indicated. Based upon the data presented below, the projected daily dose for both human and veterinary use will be from about 25 to about 200 milligrams/kilogram per day, and more usually, from about 50 to about 100 milligrams/kilogram per day.
The ability of the compounds of this invention to inhibit plasminogen activator inhibitor-1 was established by the following experimental procedures:
Test compounds are dissolved in DMSO at a final concentration of 10 mM, then diluted 100xc3x97in physiologic buffer. The inhibitory assay is initiated by the addition of the test compound (1-100 xcexcM final concentration, maximum DMSO concentration of 0.2%) in a pH 6.6 buffer containing 140 nM recombinant human plasminogen activator inhibitor-1 (PAI-1; Molecular Innovations, Royal Oak, Mich.). Following a 1 hour incubation at room temperature, 70 nM of recombinant human tissue plasminogen activator (tPA) is added, and the combination of the test compound, PAI-1 and tPA is incubated for an additional 30 minutes. Following the second incubation, Spectrozyme-tPA (American Diagnostica, Greenwich, Conn.), a chromogenic substrate for tPA, is added and absorbance read at 405 nm at 0 and 60 minutes. Relative PAI-1 inhibition is equal to the residual tPA activity in the presence of the test compounds and PAI-1. Control treatments include the complete inhibition of tPA by PAI-1 at the molar ratio employed (2:1), and the absence of any effect of the test compound on tPA alone.
This assay is based upon the non-SDS dissociable interaction between tPA and active PAI-1. Assay plates are initially coated with human tPA (10 xcexcg/ml). Test compounds are dissolved in DMSO at 10 mM, then diluted with physiologic buffer (pH 7.5) to a final concentration of 1-50 xcexcM. The test compounds are incubated with human PAI-1 (50 ng/ml) for 15 minutes at room temperature. The tPA-coated plate is washed with a solution of 0.05% Tween 20 and 0.1% BSA, then the plate is blocked with a solution of 3% BSA. An aliquot of the test compound/PAI-1 solution is then added to the tPA-coated plate, incubated at room temperature for 1 hour, and washed. Active PAI-1 bound to the plate is assessed by adding an aliquot of a 1:1000 dilution of the 33B8 monoclonal antibody against human PAI-1, and incubating the plate at room temperature for 1 hour (Molecular Innovations, Royal Oak, Mich.). The plate is again washed, and a solution of goat anti-mouse IgG-alkaline phosphatase conjugate is added at a 1:50,000 dilution in goat serum. The plate is incubated 30 minutes at room temperature, washed, and a solution of alkaline phosphatase substrate is added. The plate is incubated 45 minutes at room temperature, and color development is determined at OD405nm. The quantitation of active PAI-1 bound to tPA at varying concentrations of the test compound is used to determine the IC50. Results are analyzed using a logarithmic best-fit equation. The assay sensitivity is 5 ng/ml of human PAI-1 as determined from a standard curve ranging from 0-100 ng/ml.
The compounds of the present invention inhibited Plasminogen Activator Inhibitor-1 as summarized in Table 1.